CAJ | 학술논문

目的：构建pcDNA3．1-Ag85A-CD226真核表达质粒，制备Ag85A-CD226 DNA疫苗，经灌胃接种并观察该疫苗对小鼠免疫功能的影响。方法采用PCR方法从CD226-PCR2．1-ToPo质粒扩增CD226基因，与pcDNA3．1-Ag85A质粒连接，构建pcDNA3．1-Ag85A-CD226真核表达质粒，经脂质体转染法瞬时转染HEK293细胞株，采用RT-PCR、Western blot、免疫荧光方法检测Ag85A-CD226基因在该细胞内的表达。进一步提取纯化pcDNA3．1-Ag85 A-CD226重组质粒，用脂质体包裹制成Ag85 A-CD226 DNA疫苗。经灌胃Ag85 A-CD226 DNA疫苗接种于小鼠，乳酸脱氢酶释放法检测NK细胞杀伤活性，ELISA法检测脾细胞培养上清细胞因子分泌水平，real-time PCR法检测肠黏膜组织内细胞因子表达水平。结果成功构建真核细胞内表达pcDNA3．1-Ag85A-CD226重组质粒，并且转染HEK293细胞株检测到Ag85 A-CD226融合蛋白分子的表达。接种Ag85 A-CD226 DNA疫苗的小鼠较对照组小鼠NK细胞杀伤活性显著升高（ P＜0．01）；脾细胞培养上清中TNF-α、IFN-γ和IL-2的分泌水平显著增高（P＜0．01），而IL-4分泌水平显著降低（P＜0．01）；肠黏膜组织内TNF-α、IFN-γ和IL-2的表达水平显著升高（P＜0．01），而IL-4表达水平未见有统计学差异（P＞0．05）。结论 Ag85A-CD226 DNA疫苗经灌胃接种可诱导小鼠全身和肠道局部Th1型免疫应答增强，其效果强于单独应用Ag85 A或CD226 DNA疫苗。
목적：구건pcDNA3．1-Ag85A-CD226진핵표체질립，제비Ag85A-CD226 DNA역묘，경관위접충병관찰해역묘대소서면역공능적영향。방법채용PCR방법종CD226-PCR2．1-ToPo질립확증CD226기인，여pcDNA3．1-Ag85A질립련접，구건pcDNA3．1-Ag85A-CD226진핵표체질립，경지질체전염법순시전염HEK293세포주，채용RT-PCR、Western blot、면역형광방법검측Ag85A-CD226기인재해세포내적표체。진일보제취순화pcDNA3．1-Ag85 A-CD226중조질립，용지질체포과제성Ag85 A-CD226 DNA역묘。경관위Ag85 A-CD226 DNA역묘접충우소서，유산탈경매석방법검측NK세포살상활성，ELISA법검측비세포배양상청세포인자분비수평，real-time PCR법검측장점막조직내세포인자표체수평。결과성공구건진핵세포내표체pcDNA3．1-Ag85A-CD226중조질립，병차전염HEK293세포주검측도Ag85 A-CD226융합단백분자적표체。접충Ag85 A-CD226 DNA역묘적소서교대조조소서NK세포살상활성현저승고（ P＜0．01）；비세포배양상청중TNF-α、IFN-γ화IL-2적분비수평현저증고（P＜0．01），이IL-4분비수평현저강저（P＜0．01）；장점막조직내TNF-α、IFN-γ화IL-2적표체수평현저승고（P＜0．01），이IL-4표체수평미견유통계학차이（P＞0．05）。결론 Ag85A-CD226 DNA역묘경관위접충가유도소서전신화장도국부Th1형면역응답증강，기효과강우단독응용Ag85 A혹CD226 DNA역묘。
Objective To construct a eukaryotic expression plasmid of pcDNA 3.1-Ag85A-CD226, and to use it as DNA vaccine then further evaluate its immunogenicity through oral administration in a mouse model.Methods The CD226-PCR2.1-ToPo plasmid was used as the template to clone CD 226 gene by PCR.The CD226 gene was then inserted into pcDNA 3.1-Ag85A plasmid to construct the recombinant plas-mid of pcDNA3.1-Ag85A-CD226.After identified by restriction enzyme analysis and sequencing , the re-combinant plasmid was transfected into HEK 293 cells by using lipofection .The expression of Ag85A-CD226 gene in HEK293 cells was detected by RT-PCR, Western blot and indirect immunofluorescence assay .The purified recombinant plasmid was used to prepare the Ag 85A-CD226 DNA vaccine by liposomal encapsula-tion.The vaccine was administered intragastrically to mice .The activities of NK cells , the cytokine levels in the supernatants of spleen cell cultures and the mRNA level of cytokines in the intestines were evaluated to analyze the immunogenicity of Ag85A-CD226 DNA vaccine.Results The Ag85A-CD226 DNA vaccine was prepared successfully .The expression of Ag85A-CD226 fusion protein was detected in HEK293 cells.The activities of NK cells from mice vaccinated with Ag 85A-CD226 DNA vaccine were higher than those from other control groups (P<0.01).The level of TNF-α, IFN-γand IL-2 in the supernatants of spleen cell cul-tures and in the intestines were significantly up-regulated in comparison with other control groups ( P <0.01).The level of IL-4 in the supernatants of spleen cell cultures was down-regulated in the experimental group (P<0.01), but the level of IL-4 in intestines showed no significant difference among the five groups (P>0.05).Conclusion The Ag85A-CD226 DNA vaccine could significantly enhance Th1 type immune responses systemically and in the intestine as in comparison with those vaccinated with single dose of Ag 85A DNA vaccine or CD226 DNA vaccine.